DE2715821A1 - CELL CULTURE REACTION VESSEL AND METHOD FOR CELL BREEDING - Google Patents

Description

DRBERC-DIPL.-ING. SCHWaHE OA. DR. SANDMAIR

P.O. Box 860245, 8000 Munich 86

Attorney file 27 829

MONSANTO COMPANY ST. LOUIS / MISSOURI / U.S.A.

Cell culture reaction vessel and method for growing cells

Living cell culture in vitro is used for a wide variety of Purposes, such as the production of virus vaccines,

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the extraction of valuable by-products of cell metabolism and the production of tissue-like fabrics for artificial organs.

Various methods have so far been developed for cell culture in vitro. Stick to a popular practice and the cells grow on the flat side of appropriately shaped stationary containers such as ordinary Petri dishes or rectangular shaped culture plates. The method with flat surfaces is also used for the devices with Plate stacks are used in which a continuous plastic sheet is arranged around a series of spaced apart panels Carriers as described in U.S. Patent 3,843,454. As a support surface for cell cultivation in monolayer culture Instead of bare glass or plastic, collage-coated glass was also used. As a three-dimensional carrier matrix A collagen-coated cellulose sponge has also been proposed for cell culture.

More information about this and other conventional cell culture techniques are in standard works such as that of Kruse and Patterson, "Tissue Culture Methods and Applications", Academic Press, New York, 1973.

The use of hollow fibers or synthetic capillaries as carrier matrix for cell culture has recently been described. Knazek reports this in Science 178 , 65-67 (1972), the specific apparatus for this process is described in U.S. Patents 3,821,087 and 3,883,393. In this device, a bundle of ultrafiltration fibers in a cylindrical shape

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at home or in a case. Essentially, the device works with the help of membrane perfusion through artificial capillaries. The extensive surface of the hollow fiber system allows selective transport through the fiber walls and enables molecular exchange processes between those flowing through the fiber interior Electricity and a bath on the outside of the fibers by simple gradient diffusion. This hollow fiber apparatus is made by Knazek in Federation Proc. _3Y. ' 1978-81 (1974) and in Exptl.Cell Res. (M, 251-4 (1974). There it is used for production of HCG hormone used from human choriocarcinoma cells. The growth rate is eleven times faster than in

2
a 75 cm monolayer flask (from Falcon). Sleeve devices of the type described by Knazek and their use in cell culture are described in American Lab., October 1974, pp. 33-38.

Regardless of the usefulness of Knazek's apparatus and method it was found in practice that the use of the bundle or sleeve arrangement with the culture medium flowing through the Longitudinal capillary membranes prevent the complete penetration of the fiber bundle with the cells and an undesirable one Gradient generated in the flow of the medium. The inability of the cells to fully penetrate the bundle of fibers leads to irregularity Distribution of cells and incomplete utilization of the fiber surface available for cell attachment. The undesirable gradient consists of uneven distribution and utilization of the liquid culture medium. When the Through the reaction vessel, the cells in the vicinity of the inlet have more nutrients available, while against the medium Outlet to metabolic products such as lactic acid in the medium.

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and undesirably affect the pH and produce other toxic effects on the cells.

The invention relates to a method and a device for cell culture in vitro, wherein longitudinally oriented hollow or solid fibers arranged in a flat layer as a matrix for cell attachment and the flow of the culture medium is practically uniform through the fiber layer and across the plane of the Longitudinal axes of the fibers is passed. By using a relatively flat fiber bed and a relatively short flow path for the medium becomes the gradient of nutrients and metabolic products compared to the previous bundle or pod arrangements significantly reduced and a more complete utilization of the fiber surface achieved. The term flat layer means a layer or bed the length and width of which are substantially greater than the thickness of the layer.

Several variations of the cell culture method are within the scope of the invention and the reaction vessels possible. The following description of the preferred embodiments, along with the appended Drawings should contribute to a better understanding:

Fig. 1 is a perspective view of an embodiment of the cell culture vessel according to the invention. Figure 2 is an exploded view of the vessel of Figure 1 showing the internal parts.

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Fig. 3 is a section along line 3-3 in Fig. 1.

Fig. 4 is an exploded view of another embodiment of the invention.

Fig. 5 is a perspective view of another embodiment of the present invention Embodiment.

In Figures 1-3, the number 10 generally designates a cell culture vessel. The vessel 10 consists of a generally rectangular, parallel, tubular housing 11 with a removable lower part 12 and upper part 13. The lower housing part 12 has parallel side walls 14 and 15, and parallel end walls 16 and 17. In the same way, the upper Housing part 13 parallel side walls 18 and 10 and parallel end walls 20 and 21. In the upper housing part 13, the lower parts of the walls 18, 19, 20 and 21 are set back so that they are flush with the circumference of the inner sides of the walls 1 * 1, 15 , 16 and 17 of the lower housing portion 12 fit, while the upper flange of the walls 18, are made 19, 20 and 21, 30 so as to provide on the upper edge of the walls of I ¹ I, 15, 16 and 17 are seated.

The two parts of the housing 11 are suitably secured with conventional fasteners such as clamps, screws or bolts 37 connected to one another with wing nuts 22 as shown, or in a similar manner. If desired, it can be for the two of them Housing parts can also be used with an adhesive seal.

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In the closed state, the parts 12 and 13 delimit a chamber 23 within the housing. The bottom 24 of the housing is practically flat, while the ceiling 25, as shown, has a generally conical shape so that the height of the chamber 23 starting from the center of the ceiling 25 decreases radially in all directions.

In the walls of the reaction vessel 10 and as a connection to the chamber 23 are the medium inlets 26 and 27 in the lower part of the housing 11 and the medium outlet 28 inserted in the middle of the upper part of the housing. The medium inlets are also equipped with a medium reservoir connected, from which the medium flows in via pumps or hydrostatic pressure (not shown). The medium outlet is connected to a container for used medium (not shown). Likewise, there is an access 29 that leads to Inoculation and sampling can be used in the upper part of the housing 11 attached. To each of the inlets 26, 27, 28 and 29 includes an adapter 30, 31, 32 or 33 for regulating the medium flow and the distribution of the medium in the reaction chamber.

A layer of longitudinal fibers 34 is created as a relatively flat, low bed in chamber 23 on a rectangular manifold plate 35 arranged. A large number of small openings 36 are practically evenly spaced from one another on the surface of the plate 35 arranged for the upward flow of the medium. These openings or perforations can be, for example, about 1 to 10 mm

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Have diameters and can accordingly be up to, for example, about 10 cm apart. The plate 35 is more suitable Mounted in the chamber 23 so that it is practically in a horizontal plane above the medium inlets 26 and 27. This mounting can be done with conventional means such as holders, flanges, adhesives or the like.

The longitudinal fibers used in the cell culture vessel 10 for cell attachment can be hollow tubes or solid fibers with diameters generally from about 100 to 1000 µm. These fibers can be made from any material that is non-toxic to the cells that is easily spun into fibers and that allows cell attachment. Suitable materials include, for example, various acrylonitrile polymers, Styrene polymers, polyionic polymers, polycarbonates, polysulfones, polycarbohydrates such as cellulose and cellulose derivatives, e.g. cellulose acetate, cellulose triacetate and cellulose propionate esters, Polypeptides such as collagen, silicone rubber polymers, fluorocarbons and similar synthetic resins. Examples of suitable ones Fibers made from these materials and methods of making them are disclosed in U.S. Patents 3,228,876, 3,583,907, 3,691,068, and 3,821,087 described

As shown in Fig. 2, the fibers can be arranged in parallel or, for example, as an ordered or randomly distributed network. The fiber bed may also contain multiple layers of superimposed individual fibers and in general the use of

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one to about 50 fiber layers possible. If desired, you can Carrier plates or spacers are inserted between individual fiber layers. These support plates or spacers can be similar to the distribution plate 35, they can also be designed as grids or similar constructions, through which the culture medium can pass. The fiber bed can also consist of a continuous fiber that is turned over in this way and folded to form a plurality of longitudinal strands or segments that extend horizontally across the chamber. and run here. A continuous fiber can also be wrapped around a grid or a carrier plate and so easily two or form more layers of fibers in the bed. If desired, the fiber bed can also be erect, oblique, folded, sinusoidal, twisted or otherwise arranged, but it must maintain a relatively flat fiber layer and a relatively short medium path and the medium flow can be practically uniformly guided through the fiber bed and transversely to the plane of the fiber longitudinal axes.

Preferably, the fiber bed has a generally square horizontal cross-section in order to ensure the even distribution of the culture medium to facilitate in all directions.

In an illustrative example of a fiber bed for a rectangular one Reaction vessels with bed dimensions of 10 cm χ 10 cm can about 300 fibers with a diameter of 3.4 χ 10 cm each are ideal placed next to each other in one layer. A reaction chamber with five such layers then has an effective

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2 fiber surface for cell attachment of about 1600 cm.

However, the reaction vessel according to the invention is not based on this special dimensions, since other arrangements are also described.

When a sample reaction vessel is in operation, cell culture medium is passed through inlets 26 and 27 into chamber 23. The medium is inoculated through inlet 29 with a starting culture of a suitable mammalian Zeil line and the culture is ^{incubated at a temperature of about 20 ° C. to 40 °} C., preferably about 35 to 37 ° C. The medium can be changed regularly during the incubation, the used medium being discharged through the outlet 28 and fresh medium being reintroduced through the inlets 26 and 27. If desired, the culture medium can be aerated by conventional means prior to introduction into the cell culture reaction vessel. Following the incubation, the desired metabolic products or by-products of cell growth can be isolated from the used medium. Samples of the macromolecular material can be taken through access 29 at any point in time during the incubation. The reaction vessel is preferably operated continuously, the inlets 26 and 27 and the outlet 28 being kept open and being adjusted to the desired flow rate of the culture medium by appropriate pumping or the hydrostatic pressure.

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The culture medium flows into the lower part of the chamber 23 under the perforated plate 35, which serves as a distributor and a causes uniform distribution of the medium and a flow directed upwards and transversely to the plane of the longitudinal fiber axes. The decrease in the height of the upper chamber part 23 with increasing distance from the medium outlet 28 supports the even outflow of the used medium above the fiber bed; thus the need for the medium flowing through the fibers is met, what leads to a greater uniformity of the flow through the chamber.

The flow transverse to the fiber bed and the relatively short path through the fiber bed together result in a more uniform one Medium flow than could be achieved in previous methods with the parallel flow through a bundle of capillaries. Further a more even distribution of cell growth on the fibers and better utilization of the available fiber surface is promoted. The arrangement of the reaction vessel according to the invention therefore also includes the disadvantages of the previous reaction vessels parallel flow, in which the cells cannot penetrate the fiber bundles sufficiently and the nutrients at the inlet are consumed while gradual nutrient depletion and undesirable metabolic accumulation take place, until the medium flow reaches the other end of the reaction chamber.

FIG. 4 shows a modified cell culture reaction vessel according to the invention with an upper and a lower housing part 12

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and 13 respectively. The medium inlets 26 and 27 and the medium outlet 28 are essentially the same as in FIGS. 1 to 3, as is the access 29 and the fiber layer 34. The metal microliter can be made of stainless steel, for example, and preferably has a pore size of about 0.5 to 10 µm. It advantageously effects an even more even distribution of the culture medium than the distribution plate 35. The fiber bed can also be inserted between two metal microfilters; in this case the lower filter 40 serves as a distributor plate, the upper filter 41 as a diffusion barrier, which prevents the cells from passing through the outlet and stops the reflux of used medium. The top filter preferably has a pore size of about 10 to 100 µm. In this embodiment, the access 29 should be below the horizontal plane of the upper filter and the ceiling 42 of the reaction chamber is preferably flat and not concave as in Figures 1-3.

In Figure 5, a cell culture reaction vessel 10 as in the Figures 1 to 3, however, additional devices are provided for ventilation during incubation. at In this embodiment, the longitudinal fiber segments are hollow and permeable to air and oxygen; the reaction chamber is equipped with gas inlet and outlet lines communicating with the interior of the hollow fibers. That's how they are The gas inlet 50 and the gas outlet 51 are opposite each other in FIG

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the housing end walls 16 and 17 attached. The gas inlet 50 allows air or oxygen to enter through a claw in end wall 16 and thence into the open ends of the hollow fibers embedded in it. Gas outlet 51 enables the removal of the exhaust gas from the other fiber ends, the are also embedded in a Sanunel piece in the end wall 17. The fresh culture medium is through the inlets 26 and introduced, the used medium is discharged through the outlet 28 as above. As in the execution of Figures 1 to If access for inoculation and sampling is possible through the opening 29, the adapters 52 and 53 enable control the flow of air or oxygen through the cell culture reaction vessel .

It will be apparent that many other modifications and changes can be made of the described embodiments of the invention without departing from the basic and novel concepts of the invention possible are. For example, overflow openings or additional access and inlet openings can be made at various suitable Places in the vessel walls are attached. In the embodiments with multiple fiber layers, suitable spacers can be used be attached to keep the respective layers at a certain distance from each other. The removable The upper and lower housing parts can have further suitable projections to ensure a watertight connection between the two Parts is possible. The fiber ends can be attached to the side or end walls of the reaction chamber, they can but also in an interchangeable claw piece with potting compound

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such as epoxy and other hardening organic adhesives that can act as a seal for the fibers. The Mediumeinlässe may additionally have small openings that are arranged around the periphery of a leading into the reaction chamber and tubing to cause radial distribution of fresh culture medium.

The cell culture reaction vessel according to the invention with a flat bed can also be assembled into multiple units. Thus, for example, a plurality of reaction vessels can be easily stacked in an incubator thus forming a cell culture system with all the advantages described herein, on a larger scale.

Metal or plastic material suitable for making a relatively rigid structure can be used to construct the reaction vessel. In general, injection-molded plastic parts or machined metal parts can be used for the reaction vessel. The use of clear plastics such as polycarbonate, polystyrene, and methyl acrylate is preferred if it is to be possible to visually observe cell growth. Stainless steel is preferred because of its suitability for steam sterilization. In general, biologically inert substances are to be used for the production of all parts of the reaction chamber that come into contact with the culture medium and the culture products.

The following examples are intended to explain the invention in more detail. The cell lines and culture media used in these examples

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They are merely examples of known lines and media, and the invention is not limited thereto. So can in the invention Methods and devices also include other common mammalian cell lines such as human lung fibroblasts (WI-38), Mieren cells from rhesus monkeys (MK-2) and cervical carcinoma cells (HeLa) or other conventional culture media such as "Eagle's basal medium "and 'Earle's" or "Hank's balanced salt solution" are used will. Likewise, the inoculation, incubation and recovery processes used in the examples are only exemplary to understand. It will be apparent to those skilled in the art that other known cell culture methods can be used with the methods and devices according to the invention can be customized.

example 1

3T3 mouse embryo fibroblasts transformed by simian virus 40

2 (SV3T3) were grown to confluence in a 75 cm Falcon tissue culture flask grown, the 10 ml of a culture medium from "Dulbecco's modified Minimum Essential Medium (MEM)" with 10? fetal calf serum. After removing the used culture medium from the flask, the cells were left for several minutes trypsinized with 2 ml of $ 0.25 trypsin in phosphate-buffered saline, then the trypsin was inactivated by dilution with a further 10 ml of the same culture medium. The cells were dispensed and then placed in a sterile syringe for inoculation of a cell culture reaction vessel.

A cell culture reaction vessel according to the invention with a flat bed was sterilized with ethylene oxide to remove the ethylene

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oxide residues washed out with sterile water and equilibrated with an initial filling with "Dulbecco's modified Minimum Essential Medium (MEM)", which contained 2% fetal calf serum. The reaction vessel was made of plexiglass methyl acrylate plastic and contained a fiber bed measuring 10.16 cm (4 inches) by 10.16 cm (4 inches) by 1.27 cm (1/2 inch) high. The bed consisted of running meters (427 ft.) Of a continuous strand of hollow capillary fiber wrapped around three open mesh nylon meshes of virtually equal thickness. The fiber strand had an outer diameter of 340 μm and was spun from Amicon XM-50 (polyvinyl chloride acrylic copolymer). A 10.16 cm (4 ") by 10.16 cm (4") by 0.3175 cm (1/8 ") thick perforated manifold plate was placed directly under the fiber bed.

The reaction vessel was filled with 10 ml of the cell suspension described above (which contained 5x10 cells per ml) inoculated and Incubated at 37 C for 5 hours. Occasional shaking was used to ensure a more even attachment of the cells to the fiber support financially. Subsequent to the inoculation, "Dulbecco's modified minimum essential medium" with 2% fetal calf serum was added pumped into the reaction vessel at a rate of 10 ml per hour and through the perforated distributor plate in after directed current is directed transversely to the horizontal plane of the fiber longitudinal axes.

With increasing cell density, the flow rate was gradually increased to a maximum of 60 ml per hour, the incubation ended after 24 days.

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After the end of the incubation, the cells were removed by dismantling the reaction chamber and thoroughly washing out their contents in normal physiological saline solution obtained. The cells that fell off during washing and the fiber bed with those still attached to the fibers adherent cells (the majority of cells) were treated with 1N NaOH for 15 hours to digest all cell growth. That Digestion product was then frozen and saved for DNA analysis to determine the amount of cells recovered.

This cell culture procedure was carried out in a similar reaction chamber repeated on the flat bed, but the total incubation time was 59 days.

For comparison, this cell culture method was used in a sleeve-shaped Cell culture vessel with parallel medium flow and a fiber surface, that corresponded to that of the flatbed container, repeatedly. About 800 individual hollow capillary fibers were made from the same material and with the same diameter as above, arranged in a tube bundle 2 cm in diameter and 20 cm in length. The culture medium was not directed across the fiber bed as in the flat bed chamber, but instead flowed from the inlet one end of the sleeve to the outlet at the other end of the sleeve. As with the flat bed reaction chamber, two separate incubations were made carried out for 24 or 59 days.

The DNA analyzes of the cells obtained in the above tests can be seen in the following table:

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Chamber-
Type incubation period
in days Cell analysis
mg of DNA *
Cell equivalent Flatbed 24 18,374 1.837 χ 10 ⁹ Flatbed 59 18.84 1.84 χ 10 ⁹ Sleeve 24 4,668 0.4668 x 10 ⁹ Sleeve 59 11,836 1.18 χ 10 ⁹

* Estimated over 10 ng DNA = 10 ⁶ cells

In addition to the faster generation of a larger amount of cells , the cells in the flatbed chamber were more uniformly distributed over the existing fiber surface than in the sleeve chamber.

Example 2

The cell culture procedures of Example 1 were repeated in a flat-bed reaction vessel according to the invention, with the fiber bed from

a) Polysulfone fiber with an outer diameter of 560 μm and a total length of 12.6 m (414 ft.) and

b. polyacrylonitrile fiber with an outside diameter of 757 µm and a total length of 89 m (293 ft.)

duration.

The cells obtained were estimated by DNA analysis and corresponded

a) 1.9 10 cells for polysulfone fibers after 31 days of incubation and

b) 2.295 χ 10 cells for polyacrylonitrile fibers after 3-4 days Incubation.

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Example 3

Young hamster kidney cells (BHK) (ATCC No. CCL10) were in cultured in a flat bed reaction vessel according to the invention. "Dulbecco's modified minimum essential medium" containing 10% fetal calf serum was used and the medium aerated during a 23 day incubation period. Cell culture results similar to those in Examples 1 and 2 were obtained.

"Dulbecco's modified Minimum Essential Medium" is a commercially available one Standard culture medium. It was sold as Microbiological Associates, Bethesda, Maryland under catalog number 11-305 Dulbecco's MEM, 4.5 g / L glucose based; see also In Vitro, Vol. 6, No. 2, pp. 89-94 (1970).

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Claims (16)

Patent claims: / iJ cell culture reaction vessel, labeled through a housing, a reaction chamber within the Housing, inlets and outlets for a culture medium, which are in communication with this chamber, a plurality of longitudinally directed Fibers arranged in a flat layer fixed in the chamber, as well as distribution devices, which the flow of the culture medium practically uniformly through the fiber layer and transverse to the plane of the longitudinal axes of the Guide fibers. 2. Cell culture reaction vessel according to claim 1, characterized characterized in that the distributor device consists of an inlet distributor plate with a plurality of smaller There is perforation and is practically parallel to the fiber layer and between the fiber layer and the medium inlet is. 3. cell culture reaction vessel according to claim 2, characterized in that the distributor plate is a metal microfilter is. 4. cell culture reaction vessel according to claim 3, characterized in that an outlet metal microfilter on the outlet side of the fiber layer and practically parallel to it. 709842/0967 _ _2Q _ ORIGINAL INSPECTED 5. cell culture reaction vessel according to claim 4, characterized in that the metal microfilter distributor plate on the inlet side has a pore size of about 0.5
to 10 µm and the metal microfilter on the outlet side is one
Has pore size of about 10 to 100 µm. 6. cell culture reaction vessel according to claim 1, characterized characterized in that access to the chamber for inoculation and sampling are available. 7. cell culture reaction vessel according to claim 1, characterized in that separate gas inlets and gas outlets communicate with the chamber. 8. cell culture reaction vessel according to claim 1, characterized characterized in that the housing cover is internally conical, whereby the height of the chamber in all directions radially from Ceiling center decreases starting, and that the outlet for the culture medium is mounted in the middle of the ceiling. 9. cell culture reaction vessel according to claim 1, characterized in that the housing consists of a removable upper and lower part and one of the housing parts
is provided with projections for a watertight connection with the other housing part. 10. cell culture reaction vessel according to claim 1, characterized in that the longitudinal fibers 709842/0967 ^ _ consist of a continuous fiber strand, which is wrapped in a practically uniform thickness around a carrier grid. 11. Cell culture reaction vessel according to claim 1, characterized characterized in that the fiber layer consists of about 1 to 50 layers of individual fibers. 12. Cell culture reaction vessel according to claim 1, characterized in that the longitudinal fibers are hollow and permeable to air and oxygen, and that the reaction chamber has gas inlet and gas outlet lines for ventilation through these hollow fiber segments. 13. Cell culture reaction vessel according to claim 1, characterized in that the housing consists of a removable There is an upper and lower part, one of which is a housing part with projections for a watertight connection with the other housing part is provided; further characterized in that the fiber layer between an upper metal microfliter with a Pore size of about 10 to 100 μm and a lower metal microfilter, which serves as a distributor and has a pore size of about 0.5 to 10 μm is inserted. 14. A method for cell culture in vitro, in which the growing cells are attached to a plurality of longitudinally directed fibers which are arranged in a cell culture reaction vessel with inlets and outlets for the culture medium , characterized in that the fibers in a flat 709842/0967 _ ₂₂ _ Layer are arranged and the culture medium is distributed so that it is practically uniformly through the fiber layer and across flows to the plane of the fiber longitudinal axes. 15. The method according to claim 14, characterized in that the distribution takes place by the culture medium must flow through an inlet manifold which has a plurality of small perforations and is practically parallel to the fiber layer and between it and the inlet for the medium is arranged. 16. The method according to claim 14, characterized that the distribution plate is a metal microfilter. 709842 / 09Θ7